Endogenous Cushing's syndrome is a classic dysfunction of the endocrine system caused by prolonged exposure to inappropriately high levels of cortisol. The condition is tumor-driven and leads to obesity, diabetes, hypertension and psychiatric dysfunction, symptoms which improve with a reduction in cortisol. Cushing's syndrome disproportionately affects females, who make up 75% of the 3,000 new cases diagnosed each year in the United States. Two drugs recently approved for Cushing's, mifepristone and pasireotide, underscore the importance of developing new therapies. Pasireotide, indicated for patients with an adrenocorticotropic hormone (ACTH)-secreting pituitary tumor (Cushing's Disease), is effective in just 25% of cases and can cause hyperglycemia and diabetes. Mifepristone blocks glucocorticoid and progesterone receptors, leading to elevated cortisol and endometrial bleeding in women. Significantly, medical therapy of hypercortisolism in Cushing's is still predominantly based on off-label use of steroidogenic enzyme inhibitors that reduce cortisol, but have dose-limiting side effects such as hepatotoxicity (ketoconazole) and accumulation of steroid precursors upstream of cortisol leading to virilization in women or, less commonly, hypertension (metyrapone). Orphagen has identified the first potent small molecule antagonists to steroidogenic factor-1 (SF-1), an orphan receptor and transcription factor that regulates adrenal steroid hormone output. SF-1 antagonists block glucocorticoid production in primary rat and human adrenal cell cultures by >80% and similarly suppress pregnenolone, the first major biosynthetic steroid and the precursor to all other adrenal steroids. The antagonists are specific to SF-1 and therefore should avoid the peripheral toxicity associated with non-specific cytochrome P450 enzyme inhibitors like ketoconazole. Moreover, the SF-1 antagonist drug class, by inhibiting more than one step of adrenal steroid synthesis, should limit or avoid side effects seen with metyrapone. The objective of this proposal is to identify a first-in-class, potent and efficacious SF-1 antagonist that suppresses glucocorticoid synthesis in vivo. In Aim 1, we will design and synthesize analogs of OR-907, a first generation SF-1 antagonist, with the goal of identifying compounds with improved aqueous solubility and in vitro metabolic stability in liver microsomes (t1/2 >3h) while preserving potency (SF-1 IC50 <50nM). In Aim 2, we will evaluate pharmacokinetic properties in mice to ensure adequate oral bioavailability (>30%) and plasma (t1/2 >2h) necessary for in vivo efficacy studies. In Aim 3 we test SF-1 antagonists in mice exposed to ACTH via an osmotic pump to model chronic stimulation of adrenal function. In addition, we conduct a preliminary assessment of gonadal function in SF-1 antagonist-treated mice. The proposed Aims create a foundation for further drug discovery efforts to identify a first-in-class SF-1 antagonist clinical candidate. In he longer-term, our goal is to develop a superior medical therapy for Cushing's syndrome based on a novel mechanism of action mediated by the SF-1 receptor.